This application claims priority from pending European Patent Application EP 99204512.0 filed on Dec. 23, 1999, the contents of which are herein incorporated by this reference.
The invention relates to the field of drug discovery, diagnosis and treatment of cancer and neurological disorders. More particularly, the invention relates to a new zinc finger protein binding to a member of the a-catenins/vinculin family and discloses that human a-catulin specifically interacts with Rho-GEF Brx/proto-Lbc.
Despite extensive knowledge relating to the multitude of cancer forms (varying in appearance from solid tumours and related metastases in distinct parts of the body to leukaemias of blood cells that circulate throughout the body, and varying from being totally benign to being aggressively malignant), effective treatment of cancer remains difficult and, in general, is restricted to three types of treatments: radiation therapy; chemotherapy; and surgical therapy. Possibilities for a more specific therapy, directed against the underlying cause of a specific cancer, or groups of cancers, are currently virtually non-existant. Extensive efforts are currently being directed at providing such specific therapies through drug discovery processes aimed at identifying candidate drugs for treatment of specific cancers, groups of cancer, or neurological disorders.
Development of cancer often starts with changes in a first cell that lead to the unrestricted development and division of that first cell into an ever dividing population of cells. These changes are often an accumulation of mutations or other alterations in key genes that occur chronologically, whereby the mutated cell population looses its original, often specialised character and acquires more and more of a cancerous nature. The normal processes of growth regulation are generally dysfunctional in the altered cells. Transcription of genes that are normally infrequently expressed in non-cancerous cells may no longer be controlled in cancerous cells.
Activation of transcription of genes by transcription factors that would otherwise be dormant in the specific cell type can, for example, lead to the typical unrestricted growth and neoplastic nature of cancer. Examples are mutations in suppressor genes that function normally by generating proteins that suppress transcriptional pathways which are no longer of use in a specialised cell. Mutated suppressor genes no longer help to keep the growth of a cell under control. Drugs directed against or intervening with the specific protein-protein or protein-DNA interactions in transcriptional and/or signalling pathways controlling cell growth, cell differentiation or development can be considered typical candidate drugs for later use in specific therapies for cancer or neurological disorders, especially when the pathways that such drugs target have gone awry, leading to unrestricted growth or aberrant differentiation of cells. In the case of xcex1N-catenin, which is mainly neurally expressed, a broader or additive interpretation in relationship to neural dysfunctions is obvious.
The cadherin superfamily represents several cadherins which function in cell-cell adhesion, morphogenesis and tissue homeostasis (Takeichi, 1991; Kemler, 1992; Suzuki, 1996). The transmembrane glycoprotein E-cadherin is the best-studied prototype of this family and has been identified as a potent suppressor of invasion (Behrens et al., 1989; Frixen et al., 1991; Vleminckx et al., 1991). Recent studies revealed proof for a tumour suppressor role of human E-cadherin, as the encoding gene behaves according to the two-hit model of Knudson (1985) in infiltrative lobular cancers (Berx et al., 1995 and 1996) and diffuse gastric cancers (Becker et al., 1994 and 1996).
Cadherins function as cell-cell adhesion molecules by homophilic interactions with other cadherin molecules, but linkage to the actin cytoskeleton is also essential. The latter is achieved by the catenins (catena means chain) (Ozawa et al., 1990; Cowin, 1994), which comprise the Armadillo proteins (e.g. xcex2-catenin, plakoglobin and p120ctn) and the vinculin-like xcex1-catenins. The Armadillo catenins are proteins, known to be associated with the cytoplasmic domain of cadherins. In turn, the xcex1-catenins link xcex2-catenin and plakoglobin to the actin cytoskeleton.
These catenins were also found to be associated with APC, a cytoplasmic tumour suppressor gene product (adenomatous polyposis coli) (Peifer, 1993; Su et al., 1993). A typical example of a signal transduction pathway (via xcex2-catenin) gone wrong and leading to development of cancer, can be found with APC. The APC protein is linked to the microtubular cytoskeleton. Moreover, in the desmosomes, plakoglobin mediates a link between desmosomal cadherins and the cytokeratin cytoskeleton via desmoplakin (Korman et al., 1989; Kowalczyk et al., 1997).
Two subtypes of xcex1-catenin have been identified, xcex1E-catenin (epithelial form) (Nagafuchi et al., 1991; Herrenknecht et al., 1991) and xcex1N-catenin (neural form) (Hirano et al., 1992). Moreover, for both subtypes two isoforms, resulting from alternative splice events, have been identified (Oda et al., 1993; Uchida et al., 1994; Rimm et al., 1994). A tissue specific distribution for either of the subtypes has been reported. The epithelial xcex1E-catenin is expressed in a wide variety of tissues, but only low levels of expression have been observed in the central nervous system (CNS) (Nagafuchi et al., 1991). In contrast, xcex1N-catenin expression is more restricted to particular tissues including the nervous system, in which it is generally expressed (Hirano et al., 1992; Uchida et al., 1994).
A new homologue of the xcex1-catenins was identified and termed xcex1-catulin (Janssens et al., 1999). The xcex1-catulin cDNA has been found to be expressed in most tissues except in neural tissues. The xcex1-catulin protein shows 25% identity with the alpha-catenins, but provides higher sequence conservation in some putative functional domains.
Recently, key regulators of cadherin-mediated adhesiveness were identifed as proteins of the small-GTPase family. This family consists of the subfamilies Ras and Rho (reviewed in Braga et al., 1999). Ras GTPases are involved in growth control and differentiation. Rho GTPases participate in cytoskeletal reorganisation, activation of kinase cascades, induction of gene transcription and DNA synthesis (reviewed by Mackay and Hall, 1998). The Rho family of GTPases consists of Rho, Rac and CDC42 molecules, showing different specific effects (reviewed in Kaibuchi et al., 1999). Rho is involved in formation of stress fibers and focal adhesions, cell morphology and cell aggregation, including cadherin functionality, cell motility, membrane ruffling, smooth muscle contraction, neurite retraction in neuronal cells and cytokinesis. Rac is involved in membrane ruffling, cell motility, actin polymerization and cadherin-mediated adhesion. Cdc42 participates in filopodia formation.
Inside the cell, these GTPases are normally found associated with GDP and therefore in an inactive state. Activation occurs upon binding to GTP, a process that is tightly regulated by activating GEFs (guanine nucleotide exchange factors) and inactivating GAPs (GTPase activating factors). These transitions between GDP-bound and GTP-bound states are important regulatory processes, as for example constitutively active Rho can induce transformation in tissue culture. Moreover, deletions in Rho-GEFs such as Lbc, Vav and Dbl activate small GTPases (reviewed by Cerione and Zheng, 1996).
For the formation of cadherin-dependent cell-cell contacts, activity of endogenous Rho and Rac is required (Braga et al., 1997). Inhibition of Rho or Rac results in removal of cadherins and other molecules involved in cell-cell adhesion (Takaishi et al., 1997). Interestingly, Rac and some Rac-specific regulatory proteins like Tiam-1 and IQGAP are found to be localized to cell-cell contact sites (Habets et al., 1994; Kuroda et al., 1998). IQGAP is thought to bind to xcex2-catenin in competition with xcex1-catenin (Kuroda et al., 1998). As IQGAP can also bind and crosslink actin filaments, it could thus replace xcex1-catenin in the adhesion complex, but at the same time render the receptors less adhesive.
An example of a signaling molecule from the Rho-GEF family is Brx/proto-Lbc. Brx (Breast cancer nuclear Receptor-binding auXilliary protein) was first identified as a protein able to bind to nuclear hormone receptors, such as the estrogen receptor (ER) (Rubino et al., 1998). It was shown that two C-terminal domains (amino acids 527-950 and 961 to 1429; FIG. 21) of Brx specifically and independently interact with the C-terminal domain of the ER (amino acids 262 to 595), also know as the ligand-binding domain. A 5.3 kb Brx transcript, consistent with the Brx cDNA, is expressed in breast cancer cell lines, normal breast and testis, while larger transcripts of about 9.5 kb are found in human ovary, placenta, heart, lung, skeletal muscle, spleen, pancreas, thymus and peripheral leukocytes. As Brx contains a Dbl-homology (DH) domain and a pleckstrin homology (PH) domain, it was proposed to be a member of the Dbl family of oncoproteins (reviewed in Cerione and Zheng, 1996). As most members of the Dbl family are reported to be exchange factors for RhoA, Brx was proposed to function as a Rho-GEF.
In addition to binding to ER, Brx was shown to bind to other nuclear hormone receptors (NHRs), such as retinoid x-receptor (RXR), peroxisome proliferator-activated receptor (PPAR) and thyroid hormone receptor (THR) (Rubino et al., 1998). After ligand binding, NHRs undergo a conformational change which allows the liganded NHR to bind to DNA and transcription factors, inducing gene activation (reviewed in Beato and Sxc3xa1nchez-Pacheco, 1996). Ligand-mediated activation of NHRs is thought to be regulated by binding of an additional set of proteins. Moreover, NHRs can be activated through signals emanating from the cell surface, such as EGF-induced signaling, suggesting that a second pathway of gene activation by NHR may involve small GTPases. In the presence of estrogen, overexpression of Brx augments reporter activity of an estrogen response element (ERE). Thus Brx, as a Rho-GEF, could be involved in the GTPase pathway to regulate NHR signaling. On the other hand, also the GTPase Cdc42 was shown to be involved in Brx-dependent augmentation of estrogen response (Rubino et al., 1998). Considering that the Brx protein has been found to be highly expressed in hormone-responsive breast epithelium, the expression has also been studied in both normal and neoplastic ovarian tissues, where it was found to be expressed equally (Miller et al., 2000).
By transfecting DNA, derived from lymphoid blast crisis tissue, into NIH3T3 cells, an oncogene called Lymphoid Blast Crisis or Lbc was cloned and found to confer tumorigenicity in nude mice (Toksoz and Williams, 1994). Lbc contains a DH and a PH domain (FIG. 21). Recently, it was shown that the onco-Lbc transcript is a chimera derived from rearrangement between chromosome 15 and chromosome 7 (Sterpetti et al., 1999). From the 3xe2x80x2 242 bp derived from chromosome 7 (bp 2863 to 3106 in FIG. 22), only the first 30 bp are coding sequence. On the non-rearranged chromosome 15, a proto-Lbc gene is present, encoding a protein with a C-terminal domain of 478 amino acid residues that are missing in the onco-Lbc product. The protein sequence of proto-Lbc is largely identical to this of Brx (FIG. 22). The proto-Lbc transcript is found in a wide variety of tissues (Toksoz and Williams, 1994; Sterpetti et al., 1999). It shows high expression in spleen and testis, and lower levels in prostate, ovary, hematopoietic cells, skeletal muscle, lung, heart and small intestine. The cell lines HeLa, MOLT4, Raji, A549, G361, HL60 and SW480 are also positive for proto-Lbc. Transcript lengths vary between 5 and 9 kb, and alternative splicing at the 5xe2x80x2 end was repeatedly detected.
Both proto- and onco-Lbc are able to promote the formation of GTP-bound RhoA, although the onco-Lbc seemed to be slightly more efficient. After transfection into NIH 3T3 cells, proto-Lbc was only weakly transforming, whereas the activity of onco-Lbc was about 15 times higher (Sterpetti et al., 1999). Deletion of the a-helical and proline-rich regions (see FIG. 21) conferred 5 times higher transforming activity to proto-Lbc, but the DH and PH domains turned out to be absolutely necessary for transformation. This suggests that the proto-Lbc-specific C-terminal domain is important in negative regulation of both oncogenic and Rho-GEF activity. The onco-Lbc protein was recently found to be responsible for Rho-induced cell-rounding after thrombin stimulation of astrocytoma cells, thus providing for the first time a link between G protein-coupled receptors and Rho-mediated cytoskeletal response (Majumdar et al., 1999). Proto-Lbc also shows this effect, albeit at lower levels. The DH domain, but not the PH domain, is necessary to obtain this effect. This confirms that the DH domain confers Rho-GEF activity. The latter activity has been demonstrated for both onco-Lbc and proto-Lbc proteins (Sterpetti et al., 1999). The PH domain is probably important for subcellular localization of the protein. Moreover, Sterpetti et al. (1999) reported that the proto-Lbc protein associates with a particulate intracellular fraction, whereas onco-Lbc is completely cytosolic. A summary of functional domains in the Brx/proto-Lbc protein is given in FIG. 21. The ER binds to a central as well as to the C-terminal region. The a-helical region could be implicated in dimerisation or protein-protein association, as a homologous region is found in caldesmon, myosin, plectin and trichohyalin. The Pro-rich sequence is a potential SH3-binding site. The invention provides evidence that xcex1-catulin is binding to the C-terminal activity-regulating domain of Brx/proto-Lbc.
One of the most intriguing discoveries in the field of cadherins and catenins is the recently described association of LEF-1 (lymphocyte enhancer-binding factor-1), an architectural transcription factor (Love et al., 1995), with xcex2-catenin (Behrens et al., 1996). The interaction between xcex2-catenin and LEF-1 leads to nuclear translocation of these two proteins, implicating a central role for xcex2-catenin in the transcriptional regulation of target genes, which can lead to tumorigeneity (Huber et al., 1996; Peifer, 1997). Among the target genes induced by the xcex2-catenin/LEF-1 complex are the myc proto-oncogene (He et al., 1998) and the cyclin D1 gene (Tetsu and McCormick, 1999).
Cadherin-catenin-cytoskeleton complexes are key elements of cell-cell adhesion and regulation of motility, the importance of nuclear signalling by catenins is gaining interest and may be critical in tumorigeneity, invasion and metastasis. However, despite the existing knowledge regarding cadherins and catenins, it was not previously known what proteins are capable of translocating catenins to the nucleus, or how catenins might exert their effect on intracellular signalling and on the transcription of genes in the cell. With the means and methods of the current invention a key step has become apparent. Moreover, through the identification of such a step in the translocation of catenins to the nucleus, it has now become possible to develop means and methods for interfering with said process in, for instance, tumorigeneity, invasion and metastasis of cells.
The invention provides access to and insight into protein-protein or protein-DNA interactions in a transcriptional pathway controlling cell growth or development throughout a wide range of cells and tissues of the body. The invention further provides means, such as nucleic acids, proteins, cells, experimental animals, and methods to identify candidate drugs, for example, for use in therapy of cancer and/or neurological disorders.
In the context of this application, xe2x80x9cnucleic acidxe2x80x9d is used to mean both RNA and DNA, in single or double-stranded fashion, as well as nucleic acid hybridising thereto is meant.
As it is used in the context of this application, xe2x80x9cHomologuexe2x80x9d means a related nucleic acid that can be found in another species.
As it is used in the context of this application, the term xe2x80x9cDerivativexe2x80x9d means a nucleic acid that has been derived by genetic modifications, such as deletions, insertions, and mutations from a distinct nucleic acid or fragment thereof.
As it is used in the context of this application, xe2x80x9cCorrespondingxe2x80x9d means having a nucleic acid sequence homology of at least 80%, more preferably of at least 90%. The sequence similarities, obtained by the BLAST algorithm (Altschul et al., 1990) are given by P-scores (the more negative, the higher the similarity), not by percentages. Nevertheless, nucleotide sequence homology can be expressed as percentages (numbers of identical nucleotides per 100 nucleotides).
The terms xe2x80x9cxcex1-catenin/vinculin familyxe2x80x9d relate to a family of proteins comprising vinculin, xcex1-catulin (VR15) and xcex1-catenins such as xcex1E-catenin and xcex1N-catenin. In this regard it should be clear that xe2x80x9cfunctional homologues of xcex1-cateninxe2x80x9d comprise other members of the xcex1-catenin/vinculin family that are not 100% identical to vinculin, xcex1-catulin, xcex1E-catenin or xcex1N-catenin, but are homologous to vinculin, xcex1-catulin, xcex1E-catenin or xcex1N-catenin and can be denominated as xe2x80x9cvinculin likexe2x80x9d or xe2x80x9cxcex1-catulin-likexe2x80x9d or xe2x80x9cxe2x80x98xcex1-catenin-likexe2x80x9d.
The invention also provides an isolated and/or recombinant nucleic acid or a functional fragment, homologue or derivative thereof, corresponding to a catenin-binding protein with function in signal transduction or gene regulatory pathways, more specifically to an isolated and/or recombinant nucleic acid or a functional fragment, homologue or derivative thereof, corresponding to, for example, a gene encoding a GTPase Exchange Factor (GEF) for Rho family members, and with nucleic acid sequence as shown in FIG. 26 (SEQ. I.D. NO. 132), being part of the Brx/proto-Lbc sequences and encoding a Rho-GEF protein or fragment thereof, said protein capable of complexing or interacting with catenin or fragments thereof.
The invention provides an isolated and/or recombinant nucleic acid or a functional fragment, homologue or derivative thereof, corresponding to a catenin-binding protein with function in signal transduction or gene regulatory pathways more specifically to an isolated and/or recombinant nucleic acid or a functional fragment, homologue or derivative thereof, corresponding to, for example, a zinc finger gene with a nucleic acid sequence as shown in FIG. 1 (SEQ. I.D. NO. 1) and encoding a zinc finger protein, or fragment thereof, said protein capable of complexing or interacting with catenin or fragments thereof.
As used in the context of this application xe2x80x9cFunctional fragmentxe2x80x9d means a nucleic acid or part thereof that is functionally or structurally related to or hybridising with a distinct nucleic acid or fragment thereof. Typical examples of such a functional fragment as provided by the invention are DNA binding elements and/or subcellular localisation signals.
For example, further characterisation of nucleic acid according to the invention revealed the presence of nucleic acid encoding protein fragments encoding Cys2His2 zinc fingers with DNA binding properties. In addition, in yet another functional fragment, a nuclear localisation signal (NLS, such as PKKRKRK) (SEQ ID NO: 151) has been found.
The invention also provides a nucleic acid according to the invention wherein said protein is capable of nuclear translocation of xcex1N-catenin but not xcex1E-catenin. Co-expression of a zinc finger protein as provided by the invention or a functional fragment thereof with particular catenins ,such as xcex1N-catenin, leads to a translocation of this catenin into the nucleus. A zinc finger protein as provided by the invention protein can, for example, be isolated in a two-hybrid screening, using human xcex1N-catenin or another catenin as a bait, and is herein also called a catenin-binding protein.